The present invention relates to a detection technique and apparatus based on a substrate regenerating biosensor for use specifically but not exclusively for the sensing of cyanide. Various techniques have been devised for the detection of cyanide and determination of its concentration and these include sensitive colorimetric methods, electrochemical analysis and techniques requiring sophisticated instrumentation such as atomic absorption spectroscopy and computer assisted pattern recognition. However, there are certain inherent difficulties in the detection and determination of cyanide with many of the procedures requiring purification or volatilization of cyanide. Furthermore, most methods suffer from a lack of specificity and problems of interference. The application of physical techniques to a method of continuous monitoring is still being sought.
Biosensors for cyanide have been investigated based on cyanide's toxicity towards the biochemical activity of cytochrome oxidase. By binding to cytochrome oxidase, which is the terminal component in the electron transport chain of mitochondria, cyanide blocks an electron transfer and consequently stops electron flow in the respiratory chain.
By monitoring the electrochemical activity of cytochrome oxidase it is possible to detect the presence of toxic compounds such as cyanide, sulphide and azide from the inhibition of current. Inhibition sensors, such as those based on cytochrome oxidase operate under conditions of an enzyme limited signal rather than a transport limited one and these sensors are therefore sensitive to changes in enzyme activity.
Unfortunately inhibition sensors are susceptible to non-specific denaturation of the enzyme as well as the specific inhibitors resulting in the signal derived from the enzyme gradually decreasing even in the absence of inhibitor and limiting the operational lifetime. Furthermore substrate utilization eventually results in operational failure or the biosensor when the substrate is exhausted.
Consequently there is a need for a method for the detection and determination of concentration or toxins such as cyanide which overcomes or at least mitigates the disadvantages of the described techniques.